Silver ion chromatography of high purity conjugated linoleic acid (CLA)

ABSTRACT

A method for providing a purified conjugated linoleic acid (CLA) is disclosed. The purified conjugated linoleic acid (CLA) is formed by separating by liquid chromatography a 9-cis, 11-trans octadecadienoic acid formed by a novel synthesis of reacting an ester of ricinoleic acid with a tosyl chloride or a mesyl chloride to form a tosylate or mesylate of an ester of ricinoleic acid, and reacting the tosylate or mesylate of an ester of ricinoleic acid with diazabicyclo-undecene. Reacting an ester of ricinoleic acid with a tosyl chloride or a mesyl chloride to form a tosylate or mesylate of an ester of ricinoleic acid, and reacting the tosylate or mesylate of an ester of ricinoleic acid with diazabicyclo-undecene forms a 9-cis, 11-trans octadecadienoic acid having a purity greater than 50%, and separating by liquid chromatography forms a 9-cis, 11-trans octadecadienoic acid having a purity greater than 90%. In one aspect, the liquid chromatography uses a strong acid macroreticular ion exchange resin. In one aspect, the liquid chromatography includes silver ion liquid chromatography.

This application is a continuation-in-part of Ser. No. 08/800,567 onFeb. 18, 1997, U.S. Pat. No. 5,892,074.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to a process for providing a high purityconjugated linoleic acid (CLA) using liquid chromatography to purify aconjugated linoleic acid (CLA) produced from a novel synthesis. In oneaspect, this invention relates to a silver ion chromatography of aconjugated linoleic acid (CLA) provided by a novel synthesis of 9-cis,11-trans octadecadienoic acid, also known as 9(Z),11(E)-octadecadienoicacid, to form a high purity conjugated linoleic acid (CLA).

2. Background

Conjugated linoleic acid (CLA) is a general term used to name positionaland geometric isomers of linoleic acid.

Linoleic acid is a straight chain carboxylic acid having double bondsbetween the ninth and tenth carbons and between the twelfth andthirteenth carbons. Linoleic acid is 9-cis, 12-cis octadecadienoic acid[9(Z),12(Z)-octadecadienoic acid]. The numbers are counted from thecarboxylic acid moiety. See Formula (1). ##STR1##

Conjugated linoleic acid (CLA) has two conjugated double bonds betweenthe ninth and the twelfth carbons or between the tenth and thirteenthcarbons, with possible cis and trans combinations. Conjugated doublebonds means two or more double bonds which alternate in an unsaturatedcompound as in 1,3 butadiene. The hydrogen atoms are on the same side ofthe molecule in the case of cis. The hydrogen atoms are on the oppositeside of the molecule in the case of trans. See Formula (2). ##STR2##

The free, naturally occurring conjugated linoleic acids (CLA) have beenpreviously isolated from fried meats and described as anticarcinogens byY. L Ha, N K. Grimm and M. W. Pariza, in Carcinogenesis, Vol. 8, No. 12,pp. 1881-1887 (1987). Since then, they have been found in some processedcheese products (Y. L. Ha, N. K. Grimm and M. W. Pariza, in J. Agric.Food Chem., Vol. 37, No. 1, pp. 75-81 (1987)).

Cook et al. in U.S. Pat. No. 5,554,646 disclose animal feeds containingCLA, or its non-toxic derivatives, e.g., such as the sodium andpotassium salts of CLA, as an additive in combination with conventionalanimal feeds or human foods. CLA makes for leaner animal mass.

INTRODUCTION TO THE INVENTION

The free acid forms of CLA may be prepared by isomerizing linoleic acid.The terms "conjugated linoleic acids" and "CLA" as used herein areintended to include 9,11-octadecadienoic acid, 10,12-octadecadienoicacid, mixtures thereof, and the non-toxic salts of the acids. Thenon-toxic salts of the free acids may be made by reacting the free acidswith a non-toxic base.

Historically, CLA was made by heating linoleic acid in the presence of abase. The term CLA (conjugated linoleic acid) refers to the prior artpreparation involving alkali cooking of linoleic acid.

A conventional method of synthesizing CLA is described in Example I.However, CLA may also be prepared from linoleic acid by the action of alinoleic acid isomerase from a harmless microorganism, such as the Rumenbacterium Butyrivibrio fibrisolvens. Harmless microorganisms in theintestinal tracts of rats and other monogastric animals may also convertlinoleic acid to CLA (S. F. Chin, J. M. Storkson, W. Liu, K. Albrightand M. W. Pariza 1994, J. Nutr., 124; 694-701).

The prior art method of producing conjugated linoleic acids (CLA) can beseen in the following Example I using starting materials of linoleicacid or safflower oil.

EXAMPLE I Synthesis of Conjugated Linoleic Acids (CLA) From LinoleicAcid/Safflower Oil

Ethylene glycol (1000 g) and 500 g potassium hydroxide (KOH) are putinto a 4-neck round bottom flask (5000 ml). The flask is equipped with amechanical stirrer, a thermometer, a reflux condenser, and a nitrogeninlet. The nitrogen to be introduced is first run through two oxygentraps.

Nitrogen is bubbled into the ethylene glycol and KOH mixture for 20minutes, and the temperature is then raised to 180° C.

1000 g of linoleic acid, corn oil, or safflower oil is then introducedinto the flask. The mixture is heated at 180° C. under an inertatmosphere for 2.5 hours.

The reaction mixture is cooled to ambient conditions, and 600 ml HCL areadded to the mixture which is stirred for 15 minutes. The pH of themixture is adjusted to pH 3. Next, 200 ml of water is added into themixture and stirred for 5 minutes. The mixture is transferred into a 4 Lseparatory funnel and extracted three times with 500 ml portions ofhexane.

The aqueous layer is drained, and the combined hexane solution isextracted with four 250-ml portions of 5% NaCl solution.

The hexane is washed 3 times with water. The hexane is transferred to aflask, and the moisture in the hexane is removed with anhydrous sodiumsulfate (Na₂ SO₄). The hexane is filtered through Whatman paper into aclean 1000 ml round bottom flask, and the hexane is removed under vacuumwith a rotoevaporator to obtain the CLA. The CLA is stored in a darkbottle under argon at -80° C. until time of use.

The CLA obtained by the practice of the described prior art methods ofpreparation typically contains two or more of the 9,11-octadecadienoicacids and/or 10-12-octadecadienoic acids and active isomers thereof.After alkali treatment, the compound may be in the free acid or saltform. The CLA is heat stable and can be used as is, or it may be driedin a solvent. The CLA is readily converted into a non-toxic salt, suchas the sodium or potassium salt, by reacting chemically equivalentamounts of the free acid with an alkali hydroxide at a pH of about 8 to9.

Theoretically, eight (8) possible geometric isomers of 9,11 and10,12-octadecadienoic acid (c9,c11; c9,t11; t9,c11; t9,t11; c10,c12;c10,t12; t10,c12; and t10,t12) would form from the isomerization ofc9,c12 octadecadienoic acid. As a result of the isomerization, only fourisomers (c9,c11; c9,t11; t10,c12; and c10,c12) would be expected.Because of double bond shifts, more isomers are produced. A total oftwelve isomers have been identified so far. However, of the fourisomers, c9,t11- and t10,c12- isomers are predominantly produced duringthe autoxidation or alkali isomerization of c9,c12-linoleic acid becauseof the co-planar characteristics of 5 carbon atoms around a conjugateddouble bond and spatial conflict of the resonance radical. The remainingtwo c,c-isomers are minor contributors as are the other isomers.

The relatively higher distribution of the t,t-isomers of 9,11- or10,12-octadecadienoic acid apparently results from the furtherstabilization of c9,t11- or t10,c12-geometric isomers, which isthermodynamically preferred, during an extended processing time or longaging period. Additionally, the t,t-isomer of 9,11- or10,12-octadecadienoic acid predominantly formed during the isomerizationof linoleic acid geometrical isomers (t9,t12-, c9,t12-, andt9,c12-octadecadienoic acid) may influence the final ratio of theisomers or the final CLA content in the samples.

Linoleic acid geometrical isomers also influence the distribution ofminor contributors (c,c-isomers of 9,11- and 10,12-, t9,c11- andc11,t12-octadecadienoic acids). The 11,13-isomer might be produced as aminor product from c9,c12-octadecadienoic acid or from its isomericforms during processing.

Conjugated linoleic acid (CLA) has long been of interest to biochemistsand nutritionists. A recent article in INFORM, Vol. 7, No. 2, Feb. 1996,published by the American Oil Chemists' Society summarizes some of thedata developed so far. The article stresses the feed use for which theproduct is currently being developed, resulting in less fat and morelean meat in animals. A number of other recent articles stress effectsin fighting cancer. In many cases, one isomer, 9(Z),11(E)-CLA, has beennamed as the active isomer, mainly because it alone is incorporated intothe phospholipids of the organism being fed CIA.

CLA has been shown to have preventive effects on breast cancer in mice.CLA is not used for humans today, mostly because it is not availableexcept in impure forms. CIA is not approved by the FDA, and impuritiescan have a detrimental influence on toxicity tests to obtain FDAapproval.

The problem with CIA, as it is available today, has been the fact thatonly a diverse mixture of isomers can be made. Conventional synthesismethods involve the isomerization of linoleic acid by potassiumhydroxide at about 200° C. This procedure yields about equal amounts ofthe 9,11- and 10,12-isomers which are almost impossible to separate. Thecontent of the preferred isomer of 9(Z),11(E)-CLA in the mix is about20-30%. All of the isomers presumed to be in the mix have beensynthesized but only by very laborious methods that are quite unsuitablefor large scale manufacture.

Heating the linoleic acid in the presence of a base such as alkali,makes the double bond move over, and it does so in a haphazard way. Thegeometry changes, and the resultant product is the 9-cis, 11-transisomer in a yield of only 23-40%.

It is an object of the present invention to provide a method forproviding a purified conjugated linoleic acid (CLA).

It is an object of the present invention to provide a method forproviding a purified conjugated linoleic acid (CLA) having a puritygreater than 95%.

It is an object of the present invention to provide a method forproviding a purified conjugated linoleic acid (CLA) produced from anovel synthesis of a 9-cis, 11-trans octadecadienoic acid, also known as9(Z),11(E)-octadecadienoic acid isomer.

It is an object of the present invention to provide a method forproviding a purified conjugated linoleic acid (CLA) produced from anovel synthesis including reacting an ester of ricinoleic acid with atosyl chloride or a mesyl chloride to form a tosylate or mesylate of anester of ricinoleic acid, and reacting said tosylate or mesylate of anester of ricinoleic acid with diazabicyclo-undecene.

These and other objects of the present invention will be described inthe detailed description of the invention which follows. These and otherobjects of the present invention will become apparent to those skilledin the art from a careful review of the detailed description and fromreference to the figures of the drawings.

SUMMARY OF THE INVENTION

The present invention includes a method for providing a purifiedconjugated linoleic acid (CLA). The purified conjugated linoleic acid(CLA) is formed by separating by liquid chromatography a 9-cis, 11-transoctadecadienoic acid formed by a novel synthesis of reacting an ester ofricinoleic acid with a tosyl chloride or a mesyl chloride to form atosylate or mesylate of an ester of ricinoleic acid, and reacting thetosylate or mesylate of an ester of ricinoleic acid withdiazabicyclo-undecene. Reacting an ester of ricinoleic acid with a tosylchloride or a mesyl chloride to form a tosylate or mesylate of an esterof ricinoleic acid, and reacting the tosylate or mesylate of an ester ofricinoleic acid with diazabicyclo-undecene forms a 9-cis, 11-transoctadecadienoic acid having a purity greater than 50%, and separating byliquid chromatography forms a 9-cis, 11-trans octadecadienoic acidhaving a purity greater than 90%.

In one aspect, the liquid chromatography uses a strong acidmacroreticular ion exchange resin.

In one aspect, the liquid chromatography includes silver ion liquidchromatography.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graphical view of a gas chromatography printout of the CLAproduced by a novel synthesis for purified CLA used in the method of thepresent invention.

FIG. 2 is a graphical view of a gas chromatography printout of thestarting material of the chemical analysis of the CLA produced by anovel synthesis for purified CLA used in the method of the presentinvention.

FIG. 3 is a graphical view of a gas chromatography printout at 30minutes into the chemical analysis of the CLA produced by a novelsynthesis for purified CLA used in the method of the present invention.

FIG. 4 is a graphical view of a gas chromatography printout at 60minutes into the chemical analysis of the CLA produced by a novelsynthesis for purified CLA used in the method of the present invention.

FIG. 5 is a graphical view of a gas chromatography printout at 90minutes into the chemical analysis of the CLA produced by a novelsynthesis for purified CLA used in the method of the present invention.

DETAILED DESCRIPTION

The process of the present invention provides a method for producing ahigh purity conjugated linoleic acid (CLA) provided by a novel synthesisof the conjugated linoleic acid (CLA). In one aspect, the process of thepresent invention provides a method for liquid chromatography of highpurity conjugated linoleic acid (CLA) provided by a novel synthesis of9-cis, 11-trans octadecadienoic acid, also known as9(Z),11(E)-octa-decadienoic acid to produce a purified conjugatedlinoleic acid (CLA) not available previously.

The process of the present invention provides a method for producing 99%pure 9-cis, 11-trans octadecadienoic acid, 9(Z), 11(E) conjugatedlinoleic acid (CLA).

The process of the present invention provides a purified 9-cis, 11-transoctadecadienoic acid formed by a novel synthesis of reacting an ester ofricinoleic acid with a tosyl chloride or a mesyl chloride to form atosylate or mesylate of an ester of ricinoleic acid, and reacting thetosylate or mesylate of an ester of ricinoleic acid withdiazabicyclo-undecene.

By "liquid chromatography" of 9-cis, 11-trans octadecadienoic acid inthe context of the process of the present invention is meant passing asolution of the acid in its ester form through a bed of solid beadscausing the isomeric esters to emerge at differing time periods.

In one aspect, the process of the present invention using liquidchromatography incorporates a specified resin and provides an ability toobtain a rapid purification of the 9-cis, 11-trans octadecadienoic acidformed by the novel synthesis of the present invention, also known ascis-9, trans-11 CLA; c9,t11 CLA; 9(Z),11(E)-octadecadienoic acid; or9(Z),11(E) CLA. The specified resin includes a strong acidmacroreticular ion exchange resin. By "strong" acid is meant a resinbearing sulfonic acid substituents.

In one aspect, the process of the present invention using liquidchromatography incorporates a strong acid macroreticular silver ionexchange. In one aspect, the process of the present invention usingliquid chromatography incorporates a strong acid macroreticular ionexchange exhaustively treated with silver ions in the form of silvernitrate. By "exhaustively treated" with silver ions in the form ofsilver nitrate is meant treated with silver nitrate solution until nomore silver ion is absorbed.

The novel synthesis produces octadecadienoic acid having 75%-79% or moreof the isomer 9-cis, 11-trans octadecadienoic acid.

My novel synthesis can be summarized as follows in this detaileddescription of the novel synthesis as used in the method of the presentinvention.

I have found that a preparation of the preferred isomer of9(Z),11(E)-CLA containing 75% of the desired isomer accompanied by the9(Z),11(Z)-isomer can be made. From this material, the desired isomercan be separated by low temperature crystallization to give 90% andhigher purity of the preferred isomer.

The following conditions are required for this novel synthesis reaction.

1. Methyl ricinoleate is made into the tosylate by reaction in pyridineas solvent. When other solvents are used and pyridine only as reagent,the reaction takes days and even then does not go to completion. I havefound the reaction goes to completion overnight with pyridine as solventat room temperature.

2. The tosylate is reacted with diazabicyclo-undecene (DBU) inacetonitrile as solvent (1 hour reflux) to give a clean completereaction. Diazabicyclononene (DBN) is more expensive, but it also works.Solvents other than acetonitrile delay completion for many hours leadingto side products and incomplete reactions. Other polar butnon-hydroxylic solvents may also be useful. Examples of such otherpolar, non-hydroxylic solvents are dimethyl formamide, dimethylsulfoxide, or chloroform. Care must be taken to remove traces of thepyridine from the previous step to avoid a substitution reaction. SeeEquations (3) and (4). ##STR3##

The method for providing a purified conjugated linoleic acid (CLA) ofthe present invention includes providing a purified conjugated linoleicacid (CLA) formed by separating by liquid chromatography a 9-cis,11-trans octadecadienoic acid formed by reacting an ester of ricinoleicacid with a tosyl chloride or a mesyl chloride to form a tosylate ormesylate of an ester of ricinoleic acid, and reacting the tosylate ormesylate of an ester of ricinoleic acid with diazabicyclo-undecene.

Reacting an ester of ricinoleic acid with a tosyl chloride or a mesylchloride to form a tosylate or mesylate of an ester of ricinoleic acid,and reacting the tosylate or mesylate of an ester of ricinoleic acidwith diazabicyclo-undecene forms a 9-cis, 11-trans octadecadienoic acidhaving a purity greater than 50% by weight, preferably greater than 70%by weight, and the separating by liquid chromatography forms a 9-cis,11-trans octadecadienoic acid having a purity greater than 90% byweight, preferably greater than 95% by weight.

In one embodiment, the method for providing a purified conjugatedlinoleic acid (CLA) of the present invention includes separating byliquid chromatography to form a 9-cis, 11-trans octadecadienoic acidhaving a purity greater than about 99%.

In one embodiment, the liquid chromatography in the process of thepresent invention uses a macroreticular ion exchange resin. Themacroreticular ion exchange resin preferably is a strong acidmacroreticular ion exchange resin, more preferably a strong acidmacroreticular ion exchange resin in its silver ion form.

In one embodiment, the liquid chromatography comprises silver ion liquidchromatography. The silver ion liquid chromatography preferably uses astrong acid macroreticular silver ion exchange resin. In one embodiment,the macroreticular silver ion exchange resin includes a silver ionexchange resin exhaustively treated with silver ions in the form ofsilver nitrate.

The specified ion exchange resin of the present invention is a SulfonicAcid Resin having a particle size of about 100-120 microns and surfacearea of about 600-700 m² /gram. The specified ion exchange resin of thepresent invention is available from Keystone Research and Pharmaceuticalin Cherry Hill. N.J. 08002 as Product Code PEB-118, S/O numberso-080027.

The preferred liquid chromatography column preparation in the process ofthe present invention is as follows.

A. Material--Sulfonic acid cation exchange resin, 100-120 micron,surface area 600-700 m² /gram.

B. Pack 5 liters of wet resin into a glass column, approximately 4 ft.×4in. ID.

C. Wash the column with 3 bed columns of tap water, 15 liters at a flowrate of 60 ml/min.

D. Wash the column with 3 bed volumes of reagent grade methanol, 15liters. The fractions must be clear.

E. Wash the column with 3 bed volumes of deionized water (DI) water 15liters. At this point the column must neutral. If it is not, continuewashing until neutral.

F. Neutralize all solvents before discarding.

G. Wash the column with 0.2M aqueous silver nitrate. (34 grams/liter).This will take about 1500 grams of silver nitrate (43 liters). The M.W.of silver nitrate is 169.89.

H. Wash until eluent is no longer acid. Then let the column equilibrateovernight.

I. The column now is washed with DI water to remove excess silvernitrate. To determine if the excess silver nitrate has been eluted,remove a 1 ml sample from each fraction, and add a few drops of asaturated sodium chloride solution. If silver nitrate is present, theliquid will turn turbid. Continue until the sample stays clear. Combineall fractions and evaporate to dryness. Save the silver nitrate forfurther use.

J. Gradually wash the column with increasing amounts of methanol/DIwater.

aa. 25% methanol in DI water, 5 liters

bb. 50% methanol in DI water, 5 liters

cc. 75% methanol in DI water, 5 liters

dd. 100% methanol, 7 liters.

K. The column is now ready to use. Wrap the column with black cloth.

The preferred liquid chromatography column purification in the processof the present invention is as follows.

A. Charge 50 grams of 75-78% of the 9(Z),11(E) Isomer, and 17% of the9(Z),11(E)-isomer. Dissolve in 200 ml of methanol at a 5:1 ratio byvolume of methanol : ester. This will be in the methyl ester form.

B. Elute with methanol, using a flow rate of 15 ml/min.

C. Check by TLC, using a solvent system of petroleum ether-ethyl ester85:15. Detect by spraying with sulfuric acid dichromate spray, andcharring at 160° C.

D. The fractions which contain the CLA must be checked by GC, using thefollowing conditions.

Description: 9(Z),11(E)-Octadecadienoic Acid

Column: 30 m Length, 0.25 mm ID, .20 m Film, F.S. Material, S P 2330Phase, Helium Carrier Gas, 20 cm/sec Linear Velocity Flow Rate. TheMake-up Gas is Nitrogen at a Flow Rate of 40 cc/min. 100:1 Split Ratio,70 cc/min Vent Flow. FID Detector: Sensitivity 10⁻¹¹ ×3. Temperature:Injector 210° C., Detector 230° C., Initial Column 180° C. Chart Speed1.0 cm/min. Test Sample: Inj. 1.0 ml Vol., 10 mg/ml Conc., HexaneSolvent. Reference Sample: Pal 1.0 ml Inj. Vol., 10 mg/ml conc., HexaneSolvent

Specifications: (1.) Must be clear, tinted color is permissible. (2.)96+% 9(Z),11(E)-isomer, >99% 9,11-CLA.

E. Combine the fractions which are 91-98% pure. When using fractionswith less than 98% purity, the impurities must have shorter retentiontimes in reference to the 9(Z),11(E)-isomer. Evaporate the combinedfractions, under vacuum and no higher temperature than 35° C.

The preferred conversion to acid in the process of the present inventionis as follows.

A. If 99% pure, convert to acid. Use one mole of ester to 2 mole ofpotassium hydroxide. M.W. 9(E), 11(z)-18:2=280.45 M.W. potassiumhydroxide=56.11 Concentration=3.3 ml/gram. Dissolve the potassiumhydroxide in DI water, and then add the methanol. Reflux for 20-30minutes, use no higher heat than 65° C., under nitrogen. Check by TLCfor completion.

B. When complete, cool to room temperature. Adjust the pH to 2-4, byusing 6N HCl.

C. Add an equal amount of DI water, and extract (2) times with twice (2)the amount of hexane. Combine all hexane extracts and wash with DIwater. Remove the hexane layer and dry over sodium sulfate.

D. Check by TLC

The process of the present invention initially requires the novelsynthesis of the present invention as performed in Example II for thepreparation of the preferred isomer of 9(Z),11(E)-CLA containing 75% ofthe desired isomer accompanied by the 9(Z),11(Z)-isomer.

EXAMPLE II

12 g (0.0384 mol) methyl ricinoleate were dissolved in 30 ml pyridine,and 10.5 g tosyl chloride were added. The mixture was left overnight atroom temperature. An abundance of crystals were observed. 200 ml waterand 150 ml hexane were added. The hexane layer was washed with 2×100 mldilute acetic acid, 3×100 ml water, 1×100 ml brine, and then it wasdried and evaporated: 16.8 g (100%) tosylate.

Dissolved in 70 ml acetonitrile, and 11.7 g (0.0768 mol) DBU(diazabicycloundecene) were added. After 3 hours at room temperature,20-30% was reacted (TLC). After 15 hours, about 60% was reacted. Heatedto 75-80° C. for 30 minutes, 90% reacted. After 2 hours at 75-80° C.,all reacted and worked up. Poured into water, extracted with hexane (100ml), washed with dilute acetic acid, water, and brine, evaporated: 9.0 gslightly yellow oil.

Repeated tosylate, worked up the same way, reacted with DBU as above for11 days at approximately 12° C. All reacted from starting material. GasChromatography (GC) shows 78.51% 9(Z),11(E)-CLA and 17.14%9(Z),11(Z)-CLA 10.36 g (91%) yield. Identifications of the isomers wereconfirmed by independent University analyses, using standards obtainedfrom bacterial formation of CLA.

The gas chromatography printout from the product of Example II is shownin FIG. 1.

Example II shows the reaction of methyl ricinoleate tosylate with DBU inacetonitrile. In the first step, only tosyl chloride was used. It ispreferred for the elimination purpose. Iodide can also be used. However,oxygen is excluded since traces of elemental iodine can isomerize doublebonds which is undesirable in the novel synthesis as used in the methodof the present invention. Methane sulfonyl chloride also can be used. Inthe first step, pyridine is preferred as solvent and as base toneutralize the HCl produced in the reaction. Other solvents work muchless completely and much more slowly.

In the second step, DBN or DBU is essential. Other bases have notworked. Particularly important is the use of bases that cannot causesubstitution. Sodium hydroxide has been used previously on the chloride,not the tosylate, but a mix of isomers results in which the trans, trans[9(E),11(E)] isomer was the only one isolated in pure form.

The preferred solvent for the second step is acetonitrile, althoughothers, such as THF and toluene, also work but require highertemperatures and/or longer reaction times.

The mechanism is E2 elimination mechanism for the most part. Pure E2would require exclusive formation of the 9(Z),11(E)-isomer. Finding 17%of the 9(Z),11(Z)-isomer shows that about 34% of the reaction forms atfirst a carbocation which equilibrates and leads to 17%9(Z),11(Z)-isomer and 78% 9(Z), 11(E) isomer.

In accordance with the present invention, I have produced the cis-9,trans-11 isomer [9(Z),11(E)-isomer] and have verified independently thatthe main (78%) product is indeed the cis-9, trans-11 isomer[9(Z),11(E)-isomer], and that the minor (17%) product is the cis-9,cis-11-isomer [9(Z),11(E)-isomer]. Independent University analyses haveidentified these two peaks in gas chromatography (GC) traces. Theindependent University analyses used standards from bacterial producedCLA.

I also have found and shown a chemical proof.

Both compounds have two double bonds. I used a reaction which reducesone double bond at a time.

I then identified the three compounds with one double bond each and thecompound with no more double bonds (stearic acid ester). The reactionwas developed for unconjugated double bonds, and I have found it worksas well with the conjugated double bonds.

FIG. 2 shows the starting material mixture (GC trace). The peak at10.616 is the cis-9, trans-11 isomer. The smaller peak at 11.122 is thecis-9, cis-11 isomer. On reducing one double bond and then the other,the following reactions will take place. ##STR4##

Hence as the reaction proceeds, twice as much cis-9 ester is formed asthe others together, and since the starting material has five times asmuch cis-9, trans-11 than cis-9, cis-11, more trans-11 is formed thancis-11. Eventually, stearic ester predominates.

The chemical proof is performed and shown in Example III.

EXAMPLE III

550 mg of a CLA sample which by GC was 74% methyl9(Z),11(E)-octadecadienoate and 25% methyl 9(Z),11(Z)-octadecadienoatewere dissolved in 70 ml ethanol in a 250 ml three-neck flask equippedwith thermometer and gas inlet and outlet tubes. A slow stream of oxygenwas passed over the liquid which was heated to 40° C. with a heatingmantle. One ml of 95% hydrazine was added and the temperature went to45° C. and stayed there. Samples of 20 ml each were taken at 30 minutesand 60 minutes and the reaction stopped at 90 minutes. The samples wereacidified with concentrated HCl and the solvents evaporated, 10 ml wateradded and extracted with 10 ml hexane. Hexane solutions were used for GCdeterminations.

FIG. 3 shows the reaction mixture after 30 minutes. Both startingmaterials are reduced in content, some stearic ester has been formed,and among the products, cis-9 (also called oleate) predominates. Thereis more trans-11 (also called trans vaccenate) than cis-11 (also calledvaccenate). At 60 minutes (FIG. 4) and 90 minutes (FIG. 5), an analogouspicture is seen; the starting materials are further diminished, andstearic ester becomes the biggest peak. For comparison, the traces forpure oleate, vaccenate, and trans vaccenate were run also. The GCtechnique does not always give absolute reproducibility, and hence purecompounds are done at the same time as the mixtures. The samples wererun again on another column which pulled the peaks farther apart withsimilar results.

My novel synthesis produces octadecadienoic acid not by cooking thelinoleic acid in base, but by eliminating water from an ester ofricinoleic acid.

I have been able to recrystallize the product acid at low temperaturefrom ethyl ether and petroleum ether. After three recrystallizations, a97% pure sample results which melts at 19-20° C.

The recrystallized product purity can be verified by the methodaccording to which the acid has been made in the prior art as describedby the very laborious method of Gunstone and Russell (Chem. Soc., pp.3782, 3787, 1955). These authors report a m.p. of 19-20.2° C.

A further development of my method avoids the use of pyridine andproceeds in one step from ricinoleate. Tosylation is done withp-toluenesulfonic anhydride, thereby avoiding any chloride in the mixwhich tends to produce substitution. The base is two moles of DBU. Thesolution contains the product and the salt of DBU and p-toluenesulfonicacid. This allows the easy regeneration of DBU and, if needed, that ofp-toluene-sulfonic acid. This reaction works well with castor oilitself. The product is a triglyceride with 90% CLA since castor oil is atriglyceride with 90% ricinoleic acid and 10% of a mix of oleic,linoleic, and the like acids.

Early work in the development of the novel synthesis of the presentinvention used methyl ricinoleate and phosphorylchloride yielded12-chloro-ricinoleate. The 12-chloro-ricinoleate reacted with aqueousbase or diazabicyclononene (DBN) yielded mixtures of CLA isomers.Accordingly, it was found that although phosphorylchloride was expectedto be workable, it did not work. Chloride substitution of the hydroxylwas observed.

The reaction of the tosylate of methyl ricinoleate with reagentscontaining nucleophiles such as chloride ion, pyridine (or othernucleophilic amine) produced substitution rather than elimination.

Methyl ricinoleate reacted with Burgess' reagent, reputed to be asure-fire reagent for splitting off water, showed no likely product bygas chromatography. Moreover, Burgess' reagent would be too expensivefor a practical method.

The following actual examples describe the development of the novelsynthesis of the present invention in detail.

EXAMPLE IV

A. 1 g of ricinoleic acid methyl ester was refluxed in 30 ml acetic acidwith 3 ml acetic anhydride added and 1 g of Amberlyst-15 as catalyst for3 hours. Poured into 200 ml water and stirred for 30 minutes. Picked upin ether, ether layer washed with water and dried and evaporated. TLCshows new spot where oleate shows positive impurities and some startingmaterial.

B. 1 g of methyl ricinoleate was treated with 3 ml acetic anhydride in10 ml pyridine overnight. Poured into water and extracted with ether.TLC shows single spot higher than starting material (the acetate ofricinoleate). Product was refluxed for 5 hours in 50 ml toluene with 1 gAmberlyst-15 as catalyst.

Evaporation after washing with water and adding of ether gave a clearoil with the same spot as A (cleaner reaction). Product of A+B waspassed through silica column with hexane and "oleate" spot obtainedpure: 800 ma. Gas chromatography showed bizarre mixture.

Amberlyst-15 is a sulfonic acid resin. Acid catalyzed splitting off ofacetic acid from the acetate of ricinoleate produced complex mixtures.

EXAMPLE V

To 15.6 g (0.05 mol) of methyl ricinoleate and 5 g (0.06 mol) pyridinein 100 ml methylene chloride was added 9.5 g (0.05 mol) p-toluenesulfonyl chloride. The solution remained clear, and the next morningvery little reaction was seen by TLC (ether:pet ether 30:70). Afteranother 24 hours, very little more reaction. Added 10 ml triethyl amineand 4 g more toluene sulfonyl chloride. Left standing over the weekend.Very red solution, some solid. Washed with water, 2 N HCl and 2×water.Dried and evaporated. Picked up in 200 ml hexane, and silica added tillall the red color had been absorbed. Filtered and evaporated. TLC showeda little starting material, some almost at solvent front and mostproduct a little ahead of starting material.

The 17 g product was dissolved in 100 ml methylene chloride and 7 g DBN(diazabicyclononene) added. After 12 hours, some increase in the topspot. After 5 days, about 50% was reacted. More DBN (2 g) was added andleft standing for 2 weeks. Almost 90% top spot. Isolated bychromatography.

Tosylation with pyridine or triethylamine and tosyl chloride when heatedleads to many side products because of substitution reactions of theamines with the intermediate tosylate.

Example V shows an attempt to split off toluene sulfonic acid to makeCLA.

The Example shows a partially successful preparation of the tosylate ofmethyl ricinoleate. The reaction was slow and incomplete, use ofinappropriate solvent. The reaction product was reacted with DBN (ananalog of the later used DBU). The right product was formed butincompletely. Wrong solvent again.

The reaction to make the toluene sulfonate intermediate does not go wellin solvents other than pure pyridine. The reaction to split off thetoluene sulfonate does not go well except in acetonitrile.

It was tried to get the toluene sulfonate intermediate with triethylamine in acetonitrile. Even after a week at room temperature, thereaction was not complete. Heating is not advised since the chloride ionpresent will lead to substitution by this ion.

EXAMPLE VI

15.6 g methyl ricinoleate (0.05 mol) were dissolved in 30 ml pyridineand with ice cooling. 7 g (0.046 mol) phosphorus oxychloride were addeddrop by drop over 30 minutes. Left at room temperature for 24 hours andthen heated at 55° C. for 1 hour. Poured into a mixture of 100 ml waterand 100 ml methylene chloride. Organic layer washed with 2×200 ml 1 NHCl and 3×100 ml water. Dried and filtered and evaporated. TLC showsfast moving spot which could be the CLA product and some startingmaterial.

Example VI shows a reaction of methyl ricinoleate with phosphorusoxychloride and pyridine. The product was shown later by gaschromatography to be 12-chloro-analog of methyl ricinoleate. Thatproduct tended to form whenever chloride ions were present. Substitutionis favored over the desired elimination. Heating the toluene sulfonatein pyridine with pyridine hydrochloride present produced only the12-chloro compound.

EXAMPLE VII

1.3 g methyl ricinoleate was dissolved in 15 ml acetonitrile, heated toabout 50° C., and 1 g of Burgess salt ofmethoxycarbonylsulfamoyl)-triethyl ammonium hydroxide was added(equimolar amount). Refluxed for one hour. Poured into water andextracted with methylene chloride. TLC (ether:pet ether 30:70) showedmajor spot almost at solvent front. GC showed no methyl linoleate.

Example VII shows an attempt to dehydrate methyl ricinoleate withBurgess' salt. The reaction with Burgess' salt worked partially, rightsolvent. But many side products were also formed and Burgess' salt istoo expensive for a practical process.

This Example VII produced the desired CLA since it was later found thatthe CLA isomers do come out of the GC later than the unconjugated methyllinoleate. However, Burgess' salt is far too expensive to provide apractical procedure at $45.70 (1996) for one g.

EXAMPLE VIII

Methyl ricinoleate was prepared and converted to 12-chloro oleate bythionyl chloride.

Methyl 12-chloro oleate was treated with sodium hydroxide.

12-chloro compound was treated with DBN in methylene chloride over 3days. The result was negative. The wrong spot gets bigger, but not bymuch.

Example VIII shows an attempt to split off HCl from methyl 12-chlorooleate. Methyl 12-chloro-oleate was prepared in an attempt to work witha chloro-group instead of the hydroxy group of the ricinoleate. Attemptsto eliminate the chloro-group with DBN were unsuccessful. The TLCanalytical technique does not distinguish well between the chloro- andthe product compound. Also, reaction too slow.

The reaction of the chloro-compound with sodium hydroxide gives a mixlike the prior art method.

EXAMPLE IX

10 g (0.032 mol) methyl ricinoleate were dissolved in 60 ml pyridine and9.12 g (0.048 mol) tosyl chloride added. Left at room temperatureovernight. TLC (E:PE 30:70) shows complete reaction (only spot at app.Rf=0.6). Refluxed for one hour. TLC shows complete reaction to spot atRf=0.9. Worked up by treatment with water and hexane. Hexane layerwashed with water, dried and evaporated: 6.4 g of oil, GC looks like12-chloro compound.

Example IX shows a reaction of methyl ricinoleate with toluene sulfonyl(tosyl) chloride in pyridine and subsequent refluxing.

EXAMPLE X

Same run to make the tosylate but used 60 ml hexane and 10 ml pyridine.Needed to be warmed to effect solution. overnight at room temperatureshowed only approximately 30% reaction by TLC. Evaporated down topyridine (at approximately 40° C.) and 30 ml pyridine added. Allconverted to tosylate overnight at room temperature. Worked up and halfrefluxed in 20 ml pyridine for 40 minutes. Poured into pet ether andwater. Some oil formed between pet ether layer and water layer,pyridinium salt, care had not been taken to remove all pyridine. Yieldof ester with Rf=0.9 was 1.1 g.

The other half was dissolved in acetonitrile and 4 g (0.032 mol) DBN(diazabicyclononene) added. Left at room temperature over the weekend.All reacted. Some of the same insoluble oil formed. Yield of Rf=0.9 petether soluble oil: 2.0 g.

Example X shows (1) tosylate forms easily only in pyridine as solvent.Tosylate in acetonitrile with basic ion exchange resins have been triedwithout any success. Example X shows (2) whenever chloride ions arepresent, substitution by them is preferred. Example X shows (3) thepresence of any pyridine causes substitution by pyridine yielding thepyridinium toluene sulfonate. Since DBN cannot substitute and an esterhad formed which moved to the solvent front in TLC, the elimination ofthe elements of toluene sulfonic acid takes place, and the product wasCLA.

Example X shows success in the first step. Tosylation is completewithout side products when carried at room temperature in pyridine assolvent.

The elimination to form the desired product takes place on refluxing inpyridine. A lot of side product was observed. The easily separablepyridinium salt formed because of substitution of the tosyl group bypyridine.

EXAMPLE XI

5 g (0.016 mol) methyl ricinoleate was tosylated, worked up with diluteacetic acid (to remove all pyridine), water and brine. Dried andevaporated and dissolved in 50 ml tetrahydrofuran (THF) and 4 g DIPEAadded. Left over the weekend: no reaction. Refluxed for 1 hour, nochange. Cooled and 3.4 g DBN added. Left overnight at room temperature.Not much reacted. Refluxed for 4 hours: approximately 45% was reacted.Solvent evaporated and 20 ml acetonitrile added. Refluxed for 45minutes. All converted (by TLC).

Example XI shows an attempt to do the elimination with a hindered baseof diisopropylethylamine which cannot do a substitution like pyridine.No reaction was seen. With some DBN, some reaction was observed. A morepolar solvent is needed, and the tetrahydrofuran was determined not towork. With acetonitrile, on heating, a complete reaction was quicklyachieved. The products were submitted to gas chromatography, and the cat75:20 ratio of product to side product with otherwise few side productswas seen.

Example XI shows solvents other than acetonitrile do not work at roomtemperature. Organic bases other than DBN and DBU do not eliminateproperly. DBU is less expensive but otherwise analogous to DBN.

DBN was used in toluene. It was done at reflux (120° C.) and then gave aCLA mixture. Thus, DBN or DBU use is essential for success. Use ofacetonitrile as solvent is preferred by a large margin.

EXAMPLE XII

6.0 g of methyl ester of Example II was saponified with sodium hydroxidein methanol, overnight at room temperature. The acid was isolated byacidifying and extracting. It solidified at dry ice temperature butliquefied at 1 0° C. From it, 97% (GC) pure 9(Z),11(E)-isomer acid ofm.p. 19-20° C. was isolated by recrystallizing in the dry ice chest,twice from pet ether and once from ethyl ether.

Example XII shows the preparation of the free acid from CLA methylester. Example XII shows the preparation of the free acid from the esterand the first recrystallization of the acid and proof that purity can beincreased that way.

EXAMPLE XIII

The tosylate was prepared as in Example II, starting with 12 g methylricinoleate. The tosylate was dissolved in dimethyl sulfoxide (DMSO), 30ml, and 11.7 g of diazabicyclo-undecene (DBU) was added.

After 7 hours, TLC (pet. ether: ethyl ether 70:30) showed approximately40% completion; after 20 hours, approximately 60% done. Left at roomtemperature for 7 days: approximately 95% done; two layers had formed.

The product was not soluble in DMSO. The lower, DMSO, layer was yellow.The top layer was colorless.

Poured into 100 ml water and 100 ml hexane. The hexane layer was washedwith 50 ml 1 N HCl, 100 ml water and 100 ml brine. There was a smallamount of yellow liquid between the layers which was also discarded.

The hexane layer was dried and evaporated: almost colorless oil, 9.6 g(84%). By gas chromatography, oil was 77.1% 9(Z),11(E)-CLA and 19.0%9(Z),11(Z)-CLA.

Example XIII shows the elimination of toluene sulfonic acid fromtosylated methyl ricinoleate in DMSO as solvent.

DMSO may be a less objectionable solvent than acetonitrile. Also, theseparation of the product from the reaction mixture makes workup easier.The workup described in Example XIII with water and hexane is not benecessary for large scale runs.

EXAMPLE XIV

To a solution of 243.7 g (0.781 mol) of methyl ricinoleate in 900 mlacetonitrile was added dropwise over 2 hours a solution of 77 ml (114 g,1 mol) of methane sulfonyl chloride in 192 ml acetonitrile. After 1 hourat room temperature, the resulting mixture was vacuum filtered, and mostof the solvent was removed from the filtrate by vacuum evaporation.Water (300 ml) and a 50:50 mix of hexane and ethyl ether (300 ml) wasadded, and the upper layer was separated and washed with brine (2×200ml). Dried with sodium sulfate and the solvents evaporated.

The same reaction was carried out with 257 g of methyl ricinoleate, andboth batches were combined for vacuum distillation at 5 Torr: Fractions:(1) 33.3 g (2) 70.3 g (3) 212 g (4) 35.7 g.

Fractions (1), (2), and (3) came over at 173-174° C. Fraction (4) at174-185° C. The content of fractions (1), (2), and (3) was the same:75-77% 9(Z),11(E)-CLA, 15-17% 9(Z),11(Z)-CLA. Fraction (4) hadappreciable amounts of 9(E),11(E)-CLA and was discarded. The total yieldof acceptable product for fractions (1), (2), and (3) was 315.6 g (66%).

Example XIV shows methane sulfonyl group as the leaving group instead ofthe p-toluene sulfonyl group. With some changes in the procedure,including no pyridine as solvent in the first step, this method workedas well and gave a lesser yield of an almost identical product mix:75-77% 9(Z),11(E)-CLA and 15-17% 9(Z),11(Z)-CLA.

EXAMPLE XV

A chromatography column was filled with 5 liters of a specified strongacid macroreticular ion exchange resin exhaustively treated with silverions in the form of silver nitrate. A feed of 35 g of 68% pure9(Z),11(Z)-CLA methyl ester was passed through the chromatographycolumn. In one pass, the first product out of the column was analyzed bygas chromatography and found to be 17 g of 99% pure 9(Z),11(Z)-CLAmethyl ester.

The novel synthesis as used in the method of the present invention is anovel synthesis of conjugated linoleic acid (CLA). In one aspect, thenovel synthesis as used in the method of the present invention includesa novel synthesis of a specific form of conjugated linoleic acid (CLA),a specific isomer of CLA. The specific isomer of the present inventionis cis-9, trans-11 octadienoic acid.

There are a number of prior use patents on cis-9, trans-11octadecadienoic acid. It is believed that the presumptive activeingredient is always cis-9, trans-11 octadecadienoic acid, but priorwork has never achieved more than 40-45% pure sample.

The importance of my pure preparation is not only in the raising of leananimals but also in the potential as a cancer drug. Human trials havenot been considered before a pure preparation was at hand.

The novel synthesis as used in the method of the present inventionprovides the first practical method for the preparation of9(Z),11(E)-Octadecadienoic Acid or 9(Z),11(E)-CLA in high yield.

The novel synthesis as used in the method of the present inventionproduces the preferred isomer by splitting water off from ricinoleicacid. Elimination of water produces an (E)-double bond at the 11,12position while keeping the 9,10(Z)-double bond of the ricinoleic acid.

The purified conjugated linoleic acid (CLA) of the present invention isuseful in the treatment of carcinoma in a human through the steps ofadministering to a human a therapeutically effective amount of thepurified 9-cis, 11-trans octadecadienoic acid formed by reacting anester of ricinoleic acid with a tosyl chloride or a mesyl chloride toform a tosylate or mesylate of an ester of ricinoleic acid, reacting thetosylate or mesylate of an ester of ricinoleic acid withdiazabicyclo-undecene, and purifying the synthesized 9-cis, 11-transoctadecadienoic acid using chromatography.

The purified conjugated linoleic acid (CLA) of the present invention hasa significant potency relative to other fatty acids in respect to anability to modulate tumorigenisis.

The method of the present invention provides treatment of andsuppression of diabetes in a human through the steps of administering toa human a therapeutically effective amount of 9-cis, 11-transoctadecadienoic acid formed by reacting an ester of ricinoleic acid witha tosyl chloride or a mesyl chloride to form a tosylate or mesylate ofan ester of ricinoleic acid, and reacting the tosylate or mesylate of anester of ricinoleic acid with diazabicyclo-undecene.

The method of the present invention provides treatment of andsuppression of arthritis in a human through the steps of administeringto a human a therapeutically effective amount of 9-cis, 11-transoctadecadienoic acid formed by reacting an ester of ricinoleic acid witha tosyl chloride or a mesyl chloride to form a tosylate or mesylate ofan ester of ricinoleic acid, and reacting the tosylate or mesylate of anester of ricinoleic acid with diazabicyclo-undecene.

The method of the present invention provides treatment of andsuppression of allergies and allergic reactions in a human through thesteps of administering to a human a therapeutically effective amount of9-cis, 11-trans octadecadienoic acid formed by reacting an ester ofricinoleic acid with a tosyl chloride or a mesyl chloride to form atosylate or mesylate of an ester of ricinoleic acid, and reacting thetosylate or mesylate of an ester of ricinoleic acid withdiazabicyclo-undecene.

The method of the present invention provides treatment of andsuppression of inflammation in a human through the steps ofadministering to a human a therapeutically effective amount of 9-cis,11-trans octadecadienoic acid formed by reacting an ester of ricinoleicacid with a tosyl chloride or a mesyl chloride to form a tosylate ormesylate of an ester of ricinoleic acid, and reacting the tosylate ormesylate of an ester of ricinoleic acid with diazabicyclo-undecene.

Thus, it can be seen that the present invention accomplishes all of thestated objectives.

Although the invention has been illustrated by the preceding detaileddescription, it is not intended to be construed as being limited to thespecific preferred embodiments employed therein.

Whereas particular embodiments of the invention have been describedherein above, for purposes of illustration, it will be evident to thoseskilled in the art that numerous variations of the details may be madewithout departing from the invention as defined in the appended claims.

What is claimed is:
 1. A method for providing a purified conjugatedlinoleic acid (CLA), comprising:providing a purified conjugated linoleicacid (CLA) formed by separating by liquid chromatography a 9-cis,11-trans octadecadienoic acid formed by reacting an ester of ricinoleicacid with a tosyl chloride or a mesyl chloride to form a tosylate ormesylate of an ester of ricinoleic acid, and reacting said tosylate ormesylate of an ester of ricinoleic acid with diazabicyclo-undecene.
 2. Amethod for providing a purified conjugated linoleic acid (CLA) as setforth in claim 1, wherein said reacting an ester of ricinoleic acid witha tosyl chloride or a mesyl chloride to form a tosylate or mesylate ofan ester of ricinoleic acid, and reacting said tosylate or mesylate ofan ester of ricinoleic acid with diazabicyclo-undecene forms a 9-cis,11-trans octadecadienoic acid having a purity greater than 50%, and saidseparating by liquid chromatography forms a 9-cis, 11-transoctadecadienoic acid having a purity greater than 90%.
 3. A method forproviding a purified conjugated linoleic acid (CLA) as set forth inclaim 1, wherein said reacting an ester of ricinoleic acid with a tosylchloride or a mesyl chloride to form a tosylate or mesylate of an esterof ricinoleic acid, and reacting said tosylate or mesylate of an esterof ricinoleic acid with diazabicyclo-undecene forms a 9-cis, 11-transoctadecadienoic acid having a purity greater than 70%, and saidseparating by liquid chromatography forms a 9-cis, 11-transoctadecadienoic acid having a purity greater than 90%.
 4. A method forproviding a purified conjugated linoleic acid (CLA) as set forth inclaim 1, wherein said separating by liquid chromatography forms a 9-cis,11-trans octadecadienoic acid having a purity greater than about 99%. 5.A method for providing a purified conjugated linoleic acid (CLA) as setforth in claim 1, wherein said liquid chromatography uses amacroreticular ion exchange resin.
 6. A method for providing a purifiedconjugated linoleic acid (CLA) as set forth in claim 1, wherein saidliquid chromatography comprises silver ion liquid chromatography.
 7. Amethod for providing a purified conjugated linoleic acid (CLA) as setforth in claim 2, wherein said reacting an ester of ricinoleic acid witha tosyl chloride or a mesyl chloride to form a tosylate or mesylate ofan ester of ricinoleic acid, and reacting said tosylate or mesylate ofan ester of ricinoleic acid with diazabicyclo-undecene forms a 9-cis,11-trans octadecadienoic acid having a purity greater than 50%, and saidseparating by liquid chromatography forms a 9-cis, 11-transoctadecadienoic acid having a purity greater than 95%.
 8. A method forproviding a purified conjugated linoleic acid (CLA) as set forth inclaim 3, wherein said reacting an ester of ricinoleic acid with a tosylchloride or a mesyl chloride to form a tosylate or mesylate of an esterof ricinoleic acid, and reacting said tosylate or mesylate of an esterof ricinoleic acid with diazabicyclo-undecene forms a 9-cis, 11-transoctadecadienoic acid having a purity greater than 70%, and saidseparating by liquid chromatography forms a 9-cis, 11-transoctadecadienoic acid having a purity greater than 95%.
 9. A method forproviding a purified conjugated linoleic acid (CLA) as set forth inclaim 5, wherein said macroreticular ion exchange resin comprises astrong acid macroreticular ion exchange resin.
 10. A method forproviding a purified conjugated linoleic acid (CLA) as set forth inclaim 6, wherein said silver ion liquid chromatography uses amacroreticular silver ion exchange resin.
 11. A method for providing apurified conjugated linoleic acid (CLA) as set forth in claim 10,wherein said macroreticular silver ion exchange resin comprises a strongacid macroreticular silver ion exchange resin.
 12. A method forproviding a purified conjugated linoleic acid (CLA) as set forth inclaim 9, wherein said macroreticular silver ion exchange resin comprisesa silver ion exchange resin exhaustively treated with silver ions in theform of silver nitrate.
 13. A method for providing a purified conjugatedlinoleic acid (CLA), comprising:(a) providing a 9-cis, 11-transoctadecadienoic acid formed by reacting an ester of ricinoleic acid witha tosyl chloride or a mesyl chloride to form a tosylate or mesylate ofan ester of ricinoleic acid, and reacting said tosylate or mesylate ofan ester of ricinoleic acid with diazabicyclo-undecene; and (b)providing a purified conjugated linoleic acid (CLA) formed by separatingsaid 9-cis, 11-trans octadecadienoic acid by liquid chromatography toform a purified 9-cis, 11-trans octadecadienoic acid.
 14. A method forproviding a purified conjugated linoleic acid (CLA) as set forth inclaim 13, wherein said purified 9-cis, 11-trans octadecadienoic acid hasa purity greater than 90%.
 15. A method for providing a purifiedconjugated linoleic acid (CLA) as set forth in claim 13, wherein saidliquid chromatography uses a macroreticular ion exchange resin.
 16. Amethod for providing a purified conjugated linoleic acid (CLA) as setforth in claim 13, wherein said liquid chromatography comprises silverion liquid chromatography.
 17. A method for providing a purifiedconjugated linoleic acid (CLA) as set forth in claim 15, wherein saidmacroreticular ion exchange resin comprises a strong acid macroreticularion exchange resin.
 18. A method for providing a purified conjugatedlinoleic acid (CLA) as set forth in claim 16, wherein said silver ionliquid chromatography uses a macroreticular silver ion exchange resin.19. A method for providing a purified conjugated linoleic acid (CLA) asset forth in claim 18, wherein said macroreticular silver ion exchangeresin comprises a strong acid macroreticular silver ion exchange resinexhaustively treated with silver ions in the form of silver nitrate. 20.A method for providing a purified conjugated linoleic acid (CLA),comprising:(a) providing a 9-cis, 11-trans octadecadienoic acid formedby reacting an ester of ricinoleic acid with a tosyl chloride or a mesylchloride to form a tosylate or mesylate of an ester of ricinoleic acid,and reacting said tosylate or mesylate of an ester of ricinoleic acidwith diazabicyclo-undecene; and (b) providing a purified conjugatedlinoleic acid (CLA) formed by separating said 9-cis, 11-transoctadecadienoic acid by silver ion liquid chromatography using a strongacid macroreticular silver ion exchange resin exhaustively treated withsilver ions in the form of silver nitrate to form a purified 9-cis,11-trans octadecadienoic acid having a purity greater than 95%.